3 Purification of Nucleic Acids From Eukaryotic Cells
61
must be controlled at the pH of maximal stability, particulary ifRNA is tobe
heated. All protocols for RNA isolation use RNAse inhibitors or methods
that disrupt cells and inactivate RNAses simultaneously. (A comparison of
commercially available kits and different Iabaratory protocols is given in
Liedke et al. 1994.) The RNA is than fractionated from the other cellular
macromolecules. One important point to keep in mind isthat inactivation
of RNAses by chaotropic agents is reversible, and their removal without
parallel elimination of RNAses will result in reactivation of the enzymes
and subsequent degradation of the sample. First, a number of precautions
that can be used to avoid problems with RNAses are listed. Subsequently,
two common methods are presented. The first describes a rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells. This
results in RNA ready for Northern blot or RT-PCR. A second, more laborious protocol will provide for RNA in high yield, suitable for isolation of
poly(A)+ RNA, i.e. mRNA.
• Sterile, disposable plastic ware is essentially free of RNAses and can be Precautions
used after being autoclaved. Generallaboratory plastic ware should be
rinsed with chloroform.
• Glassware and other items are filled with diethyl pyrocarbonate (DEPC)
(0.1% in water), which is a strong, but not absolute inhibitor of RNAses.
After 2 hat 37°C, the items are rinsed several times with sterile water and
then autoclaved, if possible. These treatments remove traces of DEPC,
which might otherwise modify purine residues in the RNA by carboxymethylation.
Note: DEPC is suspected tobe carcinogen.
• All solutions (including 70% ethanol) should be prepared using RNAse
free glassware and plastic ware, DEPC-treated, autoclaved water and chemieals reserved for work with RNA. Whenever possible treat all buffers
with DEPC by adding 0.2 ml DEPC to 100 ml of the solutiontobe treated.
Stir for 12 hat 37 oc. Autoclave the solution to inactivate the remaining
DEPC.
Note: DEPC reacts rapidly with amines and cannot be used in solutions,
containing buffers such as Tris. Reserve a fresh, unopened bottle of Tris
for preparation of RN Ase free solutions.
• Store and use all items tobe used for RNA isolation at a designated place.
Wear and change frequently disposable gloves throughout the purification. Proceed as fast as possible, in one session and keep solutions at 4 oc.
61
must be controlled at the pH of maximal stability, particulary ifRNA is tobe
heated. All protocols for RNA isolation use RNAse inhibitors or methods
that disrupt cells and inactivate RNAses simultaneously. (A comparison of
commercially available kits and different Iabaratory protocols is given in
Liedke et al. 1994.) The RNA is than fractionated from the other cellular
macromolecules. One important point to keep in mind isthat inactivation
of RNAses by chaotropic agents is reversible, and their removal without
parallel elimination of RNAses will result in reactivation of the enzymes
and subsequent degradation of the sample. First, a number of precautions
that can be used to avoid problems with RNAses are listed. Subsequently,
two common methods are presented. The first describes a rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells. This
results in RNA ready for Northern blot or RT-PCR. A second, more laborious protocol will provide for RNA in high yield, suitable for isolation of
poly(A)+ RNA, i.e. mRNA.
• Sterile, disposable plastic ware is essentially free of RNAses and can be Precautions
used after being autoclaved. Generallaboratory plastic ware should be
rinsed with chloroform.
• Glassware and other items are filled with diethyl pyrocarbonate (DEPC)
(0.1% in water), which is a strong, but not absolute inhibitor of RNAses.
After 2 hat 37°C, the items are rinsed several times with sterile water and
then autoclaved, if possible. These treatments remove traces of DEPC,
which might otherwise modify purine residues in the RNA by carboxymethylation.
Note: DEPC is suspected tobe carcinogen.
• All solutions (including 70% ethanol) should be prepared using RNAse
free glassware and plastic ware, DEPC-treated, autoclaved water and chemieals reserved for work with RNA. Whenever possible treat all buffers
with DEPC by adding 0.2 ml DEPC to 100 ml of the solutiontobe treated.
Stir for 12 hat 37 oc. Autoclave the solution to inactivate the remaining
DEPC.
Note: DEPC reacts rapidly with amines and cannot be used in solutions,
containing buffers such as Tris. Reserve a fresh, unopened bottle of Tris
for preparation of RN Ase free solutions.
• Store and use all items tobe used for RNA isolation at a designated place.
Wear and change frequently disposable gloves throughout the purification. Proceed as fast as possible, in one session and keep solutions at 4 oc.